This invention relates to pharmaceutical compositions that are useful for the treatment or prevention of pathological angiogenesis or conditions requiring prevention of angiogenesis. More particularly, this invention relates to the use of quaternary derivatives of steroid agonists or antagonists having improved anti-angiogenic properties.
Angiogenesis
Angiogenesis is a complex process in which capillary blood vessels grow in an ordered sequence of events (Folkman and Klagsbrun, Science 235, 442-447, 1987; Folkman and Shing, J. Biol. Chem. 267, 10931-10934, 1992). A substantial body of evidence supports the hypothesis that tumor angiogenesis is fundamental for the growth and metastasis of solid tumors (Folkman and Klagsbrun ibid., 1987; Weidner et al. Amer. J. Pathol. 143, 401-409, 1993; O""Reilly et al. Cell 79, 316-328, 1994). Indeed, the majority of clinical tumors are not even clinically detectable until after the occurrence of neovascularization, whose induction in solid tumors is mediated by one or more angiogenic factors.
Furthermore, angiogenesis is also important in a number of other pathological processes, including, but not limited to, arthritis, psoriasis, diabetic retinopathy, retinopathy of prematurity, macular degeneration, scleroderma, hemangioma, retrolental fibroplasia, abnormal capillary proliferation in hemophiliac joints, prolonged menstruation and other disorders of the female reproductive system. Thus, methods of blocking angiogenesis are clearly necessary.
The basic mechanism of angiogenesis may be outlined briefly as follows. When a new capillary sprouts from the side of a venule, endothelial cells degrade the basement membrane, migrate toward an angiogenic source, proliferate, form a lumen, join the tips of two sprouts to generate a capillary loop, and manufacture a new basement membrane (Folkman, Perspectives in Biology and Medicine, 29, 1-36, 1985).
Degradation and remodeling of the extracellular matrix (ECM) are essential processes for the mechanism of angiogenesis. In addition, ECM components synthesized by endothelial cells (i.e., collagens, laminin, thrombospondin, fibronectin and SPARC) function to regulate endothelial cell growth, migration and shape (Bischoff, Trends Cell Biol. 5, 69-744, 1995). Bovine aortic endothelial cells (BAE), while undergoing sprouting and tube formation, synthesize collagen and SPARC. It has been proposed that type I collagen may be involved in directing the migration and assembly of BAE cells (Iruela-Arispe et al. Lab. Invest. 64, 174-186, 1991).
In order to treat angiogenesis related disorders, several inhibitors of the angiogenesis mechanism are being studied, including platelet factor 4, the fumagillin derivative AGH 1470, Interferon (xcex12a, thrombospondin, angiostatic steroids, and angiostatin (Folkman ibid., 1995; O""Reilly et al., ibid., 1994). In addition, anti-estrogens have also been shown to inhibit angiogenesis (Garliardi and Collins, Cancer Res. 53, 533-535, 1993). Unfortunately, many of these inhibitors all share the property of being relatively non-specific in their effects and, therefore, potentially toxic. A more specific inhibitor would be most useful, particularly an inhibitor that would selectively block an underlying mechanism of angiogenesis without adversely affecting other physiological functions. Furthermore, many of the compounds that are now being evaluated as anti-angiogenic agents are proteins (e.g., antibodies, thrombospondin, angiostatin and platelet factor IV) which generally suffer from poor bioavailability and are readily degraded in the body. Hence, they must be administered in high doses and frequencies.
There is, thus, a widely recognized unmet need for an inhibitor of angiogenesis which specifically blocks the proliferation of vascular structures without substantially affecting other physiological processesxe2x80x94including an inhibitor of angiogenesis associated with tumor growth or progression.
Permanently Charged Steroid Hormones and Their Antagonists
Pharmaceutical therapies for breast cancer currently consists of hormonal and cytotoxic agents. Hormonal therapy was developed because, in many women, breast cancer cells have receptors for the steroid hormone estrogen. The growth of these estrogen receptor-positive cancer cells can be stimulated by estrogen. Anti-estrogen therapy attempts to reduce or stop the synthesis of estrogen or to block the action of estrogen on the cancer cell.
Among all hormonals, tamoxifen (U.S. Pat. No. 4,536,516) holds a prevalent position. Originally used as an anti-estrogen to treat breast cancer in patients with estrogen receptor-positive tumors, the drug was also found to slow the growth of breast cancer in women with estrogen receptor-negative tumors. Tamoxifen is, therefore, useful in most patients. The anti-estrogen tamoxifen is particularly effective in delaying recurrence in breast cancer patients and in the palliative treatment of advanced metastatic breast cancer. It is also useful in the treatment of gliomas and hepatomas as well as endometrial, uterine, ovarian and prostatic neoplasms (Litherland, S. et al. Cancer Treatment Reviews, 15, 183, 1988; Jordan, C., Br. J. Pharmacol., 110, 507, 1993).
Anti-estrogens, including tamoxifen, compete with estrogen for receptor sites in cancerous tissues. occupancy of the receptor site by an anti-estrogen fails to elicit the full spectrum of transcriptional actions generated by estrogens and, thus, blocks their activity. It is generally believed that estrogens function by first binding to the target cell cytosolic receptors, and then moving into the cell nucleus, where they affect DNA transcription.
Considerable effort has been invested in the development of novel tamoxifen analogs presumed to have improved therapeutic potential, by virtue of their increased selectivity as anti-estrogenic compounds (e.g., U.S. Pat. No. 4,973,755; EP 0 168,175) or their higher affinity for the estrogen receptor (WO 92/06068).
Hydrophilic compounds and particularly compounds with ionic charges (cationic or anionic) are often very poorly distributed into the CNS and brain since a lipophilic barrier (the blood-brain barrier) exists. One method for creating a permanent charge on a drug is the incorporation of a quaternary ammonium salt (nitrogen with four hydrocarbon groups attached). Tamoxifen and other anti-estrogens that contain an amino group can be quaternized (converted to a quaternary ammonium group). Such quaternization results in imparting a permanent positive charge to the parent molecule which should effectively reduce the molecule""s penetration across physiological membranes which are inherently lipophilic and resistant to penetration of ions, particularly large ions.
Several quaternary salts of tamoxifen have been prepared and described in scientific publications (Jarman et al., Anticancer Drug Design, 1, 259, 1986). When tested in vitro, these derivatives were reported not to halt the proliferation of breast tumor cell lines grown in culture. These compounds were, therefore, predicted to be of no therapeutic value in vivo.
WO 95/26720 disclosed that unexpectedly ionic derivatives of the anti-estrogen tamoxifen, which were predicted to be of no value in vivo on the basis of their lack of activity in vitro, are, in fact, more active as anticancer agents in vivo than the parent compound. This invention is applicable, in principle, to a wide variety of other anti-estrogens where adverse side-effects may be reduced or eliminated by preventing access of the drugs to the CNS.
In a study of MCF-7 human breast cancer implanted in nude mice, TMI proved to be significantly more potent than tamoxifen in its anticancer action. TMI induced tumor regression that began almost immediately upon dose initiation and which resulted in complete regression of the implanted cancer in 40% of animals tested. The parent compound, tamoxifen, merely slowed tumor growth in that study (Cancer Res. 56, 4238, 1996).
While tamoxifen and other anti-estrogens have been reported to have angiostatic activity in tumors, the mechanism of inhibition of angiogenesis is not clear (Cancer Res. 54, 5511, 1994; Cancer Res. 53, 533, 1993).
According to the present invention, it is now disclosed that permanently charged steroid agonists and antagonists are unexpectedly potent anti-angiogenic agents. It is further disclosed that permanently charged anti-estrogens may mediate their anti-angiogenic effects by inhibiting the transcription of metalloproteases, including collagenases that are required for the restructuring of the extracellular matrix.
It is also the object of this invention to provide permanently charged steroid agonists and antagonists for the clinical treatment of protracted angiogenesis and other diseases and pathological conditions involving angiogenesis. The methods of the invention will be useful with a wide variety of steroid agonists and antagonists including, but not limited to, charged derivatives of glucocorticoids, estrogens, androgens and progestins or their respective antagonists.
In a currently preferred embodiment, it is the object of this invention to provide permanently charged agonists and antagonists for the clinical treatment of protracted angiogenesis and other diseases and pathological conditions. These anti-angiogenic anti-estrogens would possess estrogen antagonist activity, and may possess partial estrogen agonist or mixed activity, but would be limited in biodistribution by being permanently charged, thereby exhibiting reduced side effects and being beneficial for clinical use. Another object of this invention and clinical benefit is the comparatively rapid elimination from circulation of these agents due to the fact that they are not sequestered in fat itissue, thereby reducing toxicity and allowing for precise control of dosing. Yet another aspect of this invention is to provide for the formulation and drug delivery of the aforementioned anti-angiogenic anti-estrogen agents.
These and other objects of the present invention are achieved by providing compositions containing as an active ingredient a pharmaceutically effective amount of a compound of the general formula I: 
wherein:
Y is a non-toxic pharmaceutically acceptable anion;
DRUG is a radical selected from the group consisting of a steroid agonist or antagonist, a mixed agonist-antagonist, and a partial agonist;
X is a direct bond or a radical selected from the group consisting of xe2x80x94Oxe2x80x94; xe2x80x94NHxe2x80x94; xe2x80x94NRxe2x80x94, wherein R is an alkyl or aryl group with less than ten carbons; xe2x80x94PO3xe2x80x94; xe2x80x94Sxe2x80x94; xe2x80x94SOxe2x80x94; and xe2x80x94SO2xe2x80x94;
R1 and R2 are the same or different and may be a radical selected from the group consisting of H, an alkyl of 1-10 carbon atoms, an arylalkyl of 7-16 carbons, and an aryl;
R3, R4 and R5 are independently a radical selected from the group consisting of a branched or unbranched, cyclic or noncyclic, alkyl of 1-10 carbon atoms; an alkyl of up to 10 carbon atoms substituted by a carboxy, hydroxy, alkoxy, halo, or nitro group; a branched or unbranched, cyclic or noncyclic arylalkyl of 7-16 carbon atoms; and an aryl;
and n is 0-12.
Y may be exemplified by, but is not limited to, the following anions: phosphate, sulfate, chloride, bromide, iodide, an alkyl or aryl sulfonate, or an organic anion such as acetate, citrate or oxalate.
A more specific case and preferred embodiment of the general formula is: 
wherein:
Y is a non-toxic pharmaceutically acceptable anion;
anti-estrogen is a radical selected from the group consisting of an estrogen antagonist, a mixed agonist-antagonist, and a partial agonist;
X is a direct bond or a radical selected from the group consisting of xe2x80x94Oxe2x80x94; xe2x80x94NHxe2x80x94; xe2x80x94NRxe2x80x94, wherein R is an alkyl or aryl group with less than ten carbons; PO3xe2x80x94; xe2x80x94Sxe2x80x94; xe2x80x94SOxe2x80x94; and xe2x80x94SO2xe2x80x94;
R1 and R2 are the same or different and may be a radical selected from the group consisting of H, an alkyl of 1-10 carbon atoms, an arylalkyl of 7-16 carbon atoms, and an aryl;
R3, R4 and R5 are independently a radical selected from the group consisting of a branched or unbranched, cyclic or noncyclic alkyl of 1-10 carbon atoms; an alkyl of up to 10 carbon atoms substituted by a carboxy, hydroxy, alkoxy, halo, or nitro group; a branched or unbranched, cyclic or noncyclic arylalkyl of 7-16 carbon atoms; and an aryl;
and n is 0-12.
A most preferred embodiment according to the present invention comprises an anti-angiogenic compound of the general formula III: 
wherein
X is a direct bond or a radical selected from the group consisting of xe2x80x94Oxe2x80x94, xe2x80x94NRxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94SOxe2x80x94, xe2x80x94SO2xe2x80x94, and xe2x80x94PO3xe2x80x94;
R, R1 and R2 are independently a radical selected from the group consisting of H, an alkyl of 1-10 carbon atoms; an aralkyl of 7-16 carbon atoms; and an aryl;
n is 0-12;
G is a cationic radical selected from the group consisting of xe2x80x94N(Rxe2x80x2)(Rxe2x80x3)(Rxe2x80x2xe2x80x3), xe2x80x94(O)N(Rxe2x80x2)(Rxe2x80x3), xe2x80x94S(Rxe2x80x2)(Rxe2x80x3), and xe2x80x94P(Rxe2x80x2)(Rxe2x80x3)(Rxe2x80x2xe2x80x3);
Rxe2x80x2 is a radical selected from the group consisting of an alkyl of 1-10 carbon atoms; an alkyl of up to 10 carbon atoms substituted by a carboxy, hydroxy, alkoxy, halo, or nitro group; a cycloalkyl of 4-8 carbon atoms; a cycloalkyl-alkyl of 5-18 carbon atoms; and an aralkyl of 7-16 carbon atoms;
Rxe2x80x3 and Rxe2x80x2xe2x80x3 are independently a radical selected from the group consisting of an alkyl of 1-7 carbon atoms and a 4- to 8-membered nitrogen containing ring;
B is a radical selected from the group consisting of an alkyl of 1-7 carbon atoms, a halogen, a nitrogen, and a moiety which is linked to the 2-position of the phenyl that is neither the phenyl linked to the same ethylene carbon as B, nor the phenyl substituted by the radical containing the permanently ionic group G, and which is selected from the group consisting of xe2x80x94CH2C(R1)(R2)xe2x80x94 and xe2x80x94CH2xe2x80x94Oxe2x80x94;
L and M are independently 0-3;
l and m are independently 1-7; and
Y is a pharmaceutically acceptable anion.